Lau et al. Cancer & (2017) 5:10 DOI 10.1186/s40170-017-0172-1

RESEARCH Open Access PKM2 is not required for colon cancer initiated by APC loss Allison N. Lau1, William J. Israelsen1,2, Jatin Roper1,3, Mark J. Sinnamon3,4,5, Larissa Georgeon3, Talya L. Dayton1, Alissandra L. Hillis1, Omer H. Yilmaz1,6, Dolores Di Vizio7, Kenneth E. Hung3* and Matthew G. Vander Heiden1,8*

Abstract Background: CancercellsexpresstheM2isoformoftheglycolyticenzymepyruvatekinase(PKM2).PKM2 expression is not required for some cancers, and PKM2 loss can promote cancer progression; however, PKM2 has been reported to be essential in other tumor contexts, including a proposed non-metabolic role in β-catenin nuclear translocation. PKM2 is expressed in colon cancers where loss of the Apc tumor suppressor results in β-catenin nuclear translocation and aberrant activation of the canonical Wnt signaling pathway. Whether PKM2 is required in this colon cancer context has not been investigated. Results: Colon tumorigenesis was induced in mice harboring conditional Apc and Pkm2 alleles, and tumor progression was monitored by serial colonoscopy. PKM2 deletion had no effect on overall survival, the number of mice that developed tumors, or the number of tumors that developed per animal. Immunohistochemical analysis demonstrated PKM2 expression in wild-type tumors and the expected loss of PKM2 expression in tumors from Pkm2 conditional mice. Loss of PKM2 resulted in M1 expression but had no effect on nuclear β-catenin staining. These findings are consistent with tumor growth and activated Wnt signaling despite PKM2 loss in this model. We also found a large fraction of human colon cancers had very low or undetectable levels of PKM2 expression. Conclusions: PKM2 is not required for Apc-deficient colon cancer or for nuclear translocation of β-catenin in Apc-null tumor cells. These findings suggest that PKM2 expression is not required for colon tumor formation or progression. Keywords: PKM2, APC, β-catenin, Colon cancer

Background for PKM2 in different cancer contexts remains controver- Pyruvate kinase is an that catalyzes the last reac- sial [4]. For instance, PKM2 has been reported to play a tion of to produce pyruvate and ATP from role in driving cancer cell proliferation via signaling phosphoenolpyruvate and ADP. The enzymatic activity functions that are distinct from a role in glycolysis [5–7], of the M2 isoform of pyruvate kinase (PKM2) is regu- including a nuclear function to activate β-catenin down- lated by growth signaling and multiple allosteric effec- stream of growth signaling [8]. However, other work has tors, coupling PKM2 activity to the metabolic and shown that PKM2 is dispensable for the growth and main- signaling state of the cell in order to promote prolifera- tenance of xenograft tumors [9], that loss of PKM2 can tive metabolism in appropriate contexts [1, 2]. PKM2 is promote progression of BRCA1 loss-driven breast cancer expressed in a wide variety of cancer types and both [10] and medulloblastoma tumor growth [11], and that metabolic and non-metabolic functions for PKM2 in PKM2 is not required for leukemia or liver tumor forma- cancer have been suggested [3]; however, the requirement tion [12, 13]. PKM2 is expressed in colon cancers [14] and has been * Correspondence: [email protected]; [email protected] reported to facilitate colon cancer cell proliferation, migra- 3Department of Medicine, Tufts Medical Center, Boston, MA 02111, USA tion, and the epithelial-mesenchymal transition [15–17]. 1Koch Institute for Integrative Cancer Research and the Department of Biology at Massachusetts Institute of Technology, Cambridge, MA 02139, USA Immunohistochemical analysis of human colon tumors Full list of author information is available at the end of the article revealed that tumors express higher levels of PKM2 as

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lau et al. Cancer & Metabolism (2017) 5:10 Page 2 of 7

compared to paired normal colon tissue and that in- tumor growth, latency, and multiplicity per animal were creased PKM2 expression is associated with greater tumor determined. In this method, a section of distal colon is stage and with lymph node metastasis [15]. Due to the transiently ligated and adenovirus-encoding Cre recom- controversy surrounding the requirement for PKM2 by tu- binase is introduced into the lumen of the colon. Cre mors and the functions of this enzyme in cancer, we tested expression in infected cells results in Apc loss and the importance of PKM2 in colon cancer by crossing mice stochastic tumor initiation in the distal colon that can be harboring a conditional Pkm2 allele [10] to mice with con- monitored with optical colonoscopy [18]. Representative CKO ditional alleles of the Apc (Apc ) tumor suppressor colonoscopy images showing colon tumors at various [18]. This genetically engineered mouse model of colon stages of progression are shown in Fig. 1b. cancer has been applied to study the role of oncogenic To verify that Pkm2 was deleted in tumors from flox/flox flox/flox Kras and Braf in tumorigenesis [19–22]. By comparing Apc ;Pkm2 mice, we analyzed genomic flox/flox +/+ flox/flox mice with and without Pkm2 conditional alleles in this DNA from Apc ;Pkm2 and Apc ;Pkm2- flox/flox model, we sought to determine whether PKM2 is essential colon tumors to confirm that PKM2 recom- for colon cancer initiation and progression following Apc bination had occurred (Fig. 2a). There was no loss. significant difference in the fraction of mice develop- ing one or more tumors 3 weeks, 6 weeks, or at Results later time points following Cre administration in flox/flox flox/flox PKM2 is expressed in many normal tissues, including Apc ;Pkm2 mice compared with control flox/flox +/+ the intestine [23], while the pyruvate kinase M1 (PKM1) Apc ;Pkm2 mice (Fig. 2b). There were also isoform, encoded by an alternative splice product of the no differences in the number of tumors arising per PKM gene, is also expressed in the intestine [14]. To mouse (Fig. 2c) over the same period of time. determine which cell types of the colon express each iso- Overall survival was not significantly different be- flox/flox +/+ flox/flox flox/flox form, we performed immunohistochemistry on normal tween Apc ;Pkm2 and Apc ;Pkm2 mouse colon tissue using PKM2 and PKM1 isoform- mice with Apc loss driven colon tumors (Fig. 2d). specific antibodies [10]. PKM2 staining was observed in We also analyzed pyruvate kinase expression in tu- the epithelial cells of the crypts, including crypt base mors isolated from these mice using immunohistochem- cells that include colon stem cells, but was not promin- istry with pyruvate kinase isoform-specific antibodies. As ent in the lamina propria (Fig. 1a). The muscularis expected, analysis of colon lesions showed that PKM2 propria layer expressed PKM1 but not PKM2 (Fig. 1a). wild-type tumors expressed almost exclusively PKM2 This makes it likely that tumors in this model arise from (Fig. 3a). Similar to breast tumors [10], some of these PKM2-expressing cells. PKM2 wild-type tumor cells appeared to express very lit- To generate Apc-deficient colon tumors, colon tissue tle PKM2 despite a lack of PKM1 expression (Fig. 3a, CKO from Apc mice with and without homozygous condi- Additional file 1: Figure S1A). PKM2 expression in tional Pkm2 alleles was exposed to adenoviral Cre, and normal colon tissue and PKM1 expression in the lamina

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Fig. 1 PKM2 is expressed in colon epithelial cells where loss of APC results in tumor formation. a FFPE colon tissue sections from mice were stained with hematoxylin and eosin (H&E) and isoform-specific antibodies against PKM2 or PKM1 as shown. Scale bars represent 50 μmforallimagesat×20 magnification (left) and 20 μm for all images at ×40 magnification (right). b Adenoviral Cre was used to induce colon tumors, with ligation-restricting adenoviral Cre exposure to the distal colon. Lesion appearance and tumor progression were monitored by serial colonoscopy as shown. Representative tumors from four different Apcflox/flox Pkm2flox/flox mice are shown at various stages of progression Lau et al. Cancer & Metabolism (2017) 5:10 Page 3 of 7

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Fig. 2 PKM2 deletion does not affect tumor penetrance, multiplicity, or survival in mice with APC deletion. a Colon tumors were initiated in Apcflox/flox Pkm2+/+ and Apcflox/flox Pkm2flox/flox mice, and PCR genotyping of the Pkm2 allele in tumors arising in each condition is shown. Analysis of tail DNA from Pkm2+/+ and Pkm2flox/flox mice is shown as a control. b Tumor penetrance in Apcflox/flox Pkm2+/+ and Apcflox/flox Pkm2flox/flox mice was determined by colonoscopy. The fraction of mice with tumors did not differ significantly between cohorts at 3 weeks post-infection, at 6 weeks post-infection, or at final inspection (n =24Pkm2flox/flox mice, 28 Pkm2+/+ mice per cohort, p > 0.1 at all time points, Fisher’sexacttest).c Apcflox/flox Pkm2flox/flox mice had similar tumor multiplicity when compared to Apcflox/flox Pkm2+/+ mice. Multiplicity is defined as the number of tumors per tumor-bearing animal (n =17Pkm2flox/flox mice, 18 Pkm2+/+ mice, p >0.25).d Kaplan-Meier curve showing survival of the Apcflox/flox Pkm2+/+ and Apcflox/flox Pkm2flox/flox cohorts. Pkm2 deletion had no statistically significant effect on survival (n =24Pkm2flox/flox mice, 28 Pkm2+/+ mice per cohort, p = 0.7060, Log-rank (Mantel-Cox) test) propria were confirmed in these same tissue sections Additional file 1: Figure S1C). These data suggest that (Additional file 1: Figure S1B). Colon tumors from PKM2 is not required for nuclear translocation of β- flox/flox flox/flox Apc ;Pkm2 mice showed loss of PKM2 catenin downstream of APC loss in colon cancer. expression in areas of the tumors; however, PKM2 ex- To examine PKM2 expression in human colon tumors, pression was maintained in some areas and in normal we performed PKM2 immunohistochemistry on 41 colon tissue (Fig. 3b). Examination of serially stained colon tumor tissue array samples. Nearly half of the sections showed that tumor areas with loss of PKM2 samples (20/41) were negative for PKM2 expression, and displayed expression of PKM1 (Fig. 3b), findings simi- 18/41 expressed low levels of PKM2 (Fig. 4a, b). The lar to what was previously observed in both tumor lack of expression of PKM2 in human colon tumors fur- and normal tissue [10, 12, 13]. ther supports a model in which PKM2 upregulation is The APC tumor suppressor protein is a negative not required and is instead dispensable for the formation regulator of canonical Wnt signaling. APC loss results in and growth of at least some colon tumors. nuclear translocation of β-catenin, and strong nuclear β-catenin staining is observed in this colon cancer model Discussion [18]. Because work in glioblastoma has suggested that Taken together, these results argue that PKM2 is not PKM2 is required for β-catenin activation downstream required for APC-deficient colon cancer. These find- of EGF stimulation in that tissue [8], we determined ings are consistent with previous results suggesting whether loss of PKM2 impacted β-catenin localization in that PKM2 is not required for BRCA1-deficient breast flox/flox flox/flox tumors arising in Apc ;Pkm2 mice (Fig. 3a, b, cancer [10], medulloblastoma [11], BCR-ABL-driven Additional file 1: Figure S1C). β-catenin staining was leukemia [12], or hepatocellular carcinomas with found only at the plasma membrane of non-transformed diverse genetics that arise in animals with metabolic colon epithelial cells (Additional file 1: Figure S1C). In syndrome [13]. While loss of PKM2 slowed leukemia contrast, both PKM2 wild-type and PKM2-null tumor progression, deletion of PKM2 increased breast cancer cells showed strong nuclear β-catenin staining (Fig. 3a, b, and medulloblastoma progression and could promote Lau et al. Cancer & Metabolism (2017) 5:10 Page 4 of 7

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Fig. 3 PKM2 expression is lost in colon tumor cells in Pkm2 conditional animals. a Tissue sections from tumors arising in Apcflox/flox Pkm2+/+ mice were stained with hematoxylin and eosin (H&E) or with antibodies against PKM2, PKM1, or β-catenin as shown. b Colon tumors arising in Apcflox/flox Pkm2flox/flox mice were stained with hematoxylin and eosin (H&E) or with antibodies against PKM2, PKM1, or β-catenin as shown. A PKM2 negative region that switches to PKM1 expression is marked with an arrowhead, and a PKM2 positive region is marked with an arrow. Scale bars represent 50 μmforallimagesat×20 magnification (top row in each panel) and 20 μm for all images at ×40 magnification (bottom row in each panel)

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Fig. 4 Human colon tumors express low levels of PKM2. a Representative TMA cores containing human colon tumors showing PKM2 staining scored as 0, 1, or 2. Scale bars represent 50 μm for all images at ×20 magnification (left) and 20 μm for all images at ×40 magnification (right). b Distribution of PKM2 staining intensities for 41 human colon tumors Lau et al. Cancer & Metabolism (2017) 5:10 Page 5 of 7

hepatocellular carcinoma. We find here that the ab- not have a unique role but instead allows cancer cells to sence of PKM2 does not prevent sporadic colon integrate metabolic and signaling inputs to support tumor initiation and progression, nor does it affect tumor growth. tumormultiplicityorsurvival.PKM2expressionwas low and variable in PKM2 wild-type tumors, as well Conclusions as in human colon tumors. An isoform switch from Our study found that PKM2 is not required for colon PKM2 to PKM1 expression was observed in PKM2- tumor initiation or growth driven by APC loss. Loss of null tumor regions, findings that are reminiscent of PKM2 in APC-deficient tumors resulted in higher PKM1 those observed in BRCA1 loss-driven breast cancer expression but did not change β-catenin expression [10] or leukemia [12] models, and argue that dispens- patterns, further arguing that PKM2 is not required for ability of PKM2 is not limited to specific tumor types β-catenin nuclear localization downstream of APC loss. or to individual driver mutations. Recent work has Many human colon tumors examined in this study also shown that PKM2 is also dispensable during normal were found to have very low or undetectable PKM2 embryonic development, further supporting the idea expression, also supporting the conclusion that PKM2 is that PKM2 is not necessary for cell proliferation in dispensable for the growth of some colon tumors. various physiological contexts [13]. Since PKM1 is constitutively active, whereas PKM2 Methods activity can change between low activity and high- Mouse models and tumor induction CKO activity states, PKM1 expression has been associated Pkm2flox/flox mice [10] were bred to the Apc mice with higher pyruvate kinase activity and decreased [18] to generate animals of relevant genotypes. proliferation [10, 24]. Although we did not measure Ad5CMVcre adenovirus was obtained from the Gene overall pyruvate kinase activity in PKM2 wild-type Transfer Vector Core, University of Iowa. Surgeries to versus PKM2-null tumors, we did observe higher PKM1 deliver Ad5CMVcre adenovirus to the colon were per- expression in PKM2-null tumors. In contrast to other formed as described previously [18]. models where PKM2 has been deleted [24, 25], PKM1 expression following PKM2 deletion in the colon did not Colonoscopy appear to affect tumor progression or proliferation. This Mice were fasted overnight and anesthetized using 2% may be related to the possibility that not all tumor cells ef- isoflurane. The colon was washed with PBS and colonos- ficiently deleted PKM2, as PKM2 expression was retained copy was performed using a custom-made system as in some areas of the tumor, likely due to the multi-clonal described previously [18]. nature of colon tumors in this model. The pattern of retained PKM2 expression is most consistent with ineffi- Immunohistochemistry cient deletion of PKM2 in some founder clones, as mixed Sections from formalin-fixed paraffin-embedded tissue cell populations of PKM2-expressing and deleted cells were stained with hematoxylin and eosin or with anti- were not observed. This argues against a selective pressure bodies against PKM2 (Cell Signaling Technologies #4053, to lose PKM2 expression in promoting tumor growth fol- 1:800 dilution), PKM1 (Cell Signaling Technologies lowing APC loss. Nevertheless, the loss of PKM2 expres- #7067, 1:1000 dilution), or β-catenin (BD Transduction sion in some tumor areas suggests that PKM2 is not Laboratories #610153, 1:100 dilution) as previously described required for tumor formation in this model. [10, 18]. We analyzed a multi-tissue tissue microarray Our results demonstrate that nuclear localization of (TMA) containing tissue obtained from the archives of the β -catenin downstream of canonical Wnt signaling is not InstituteofPathologyattheUniversityofBasel[29,30].The dependent on PKM2. PKM2 has been reported to play a TMA was scored for IHC intensity independently by both a β role in nuclear -catenin localization through a Wnt- pathologist (D.D.V.) and another member of the team independent mechanism downstream of EGF receptor (T.L.D.). Tumors that showed no positive staining for PKM2 signaling in glioblastoma [8]. Therefore, it is possible were given a score of 0, those with weak staining were given β that PKM2 could play a role in -catenin nuclear a score of 1, and tumors with strong PKM2 staining were localization driven by EGF signaling in colon cancers given a score of 2. where this pathway is hyperactive. Altered EGF receptor signaling can be a driver of colon cancer [26], and Additional file whether PKM2 has a different requirement in this context is not known. Nevertheless, these data argue Additional file 1: Figure S1. Additional immunohistochemistry images. against the requirement for a unique PKM2 activity in A) PKM2 wild-type tumors show variability in PKM2 staining; representative malignancy outside of specific contexts [6, 7, 27, 28]. images of PKM2 staining in tumors from Apcflox/flox Pkm2+/+ mice. B) PKM2 staining of normal colon and PKM1 staining of muscle fiber from the same Instead, the findings support a model where PKM2 does Lau et al. Cancer & Metabolism (2017) 5:10 Page 6 of 7

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